Fig. 4.

SK11 and SK12 phosphorylate TTG1 mainly through Ser 215. a GST-SK11 and GST-SK12 alter the electrophoretic mobility of MBP-TTG1 on a Phos-tag-containing gel (upper panel), but not on a regular SDS-PAGE gel (lower panel). GST-SK11 and GST-SK12 do not affect the electrophoretic mobility of MBP on both Phos-tag-containing and regular SDS-PAGE gels. Arrowheads and arrows indicate phosphorylated and unphosphorylated protein forms, respectively. b Bikinin treatment inhibits phosphorylation of 4HA-TTG1 in ttg1-13 g4HA-TTG1. Total protein was extracted from ttg1-13 g4HA-TTG1 siliques collected at 4 days after pollination, which were not treated (NT), mock-treated (Mock), or treated by bikinin (Bik) for 2 h. These protein extracts were analyzed by immunoblot on Phos-tag-containing (upper panel) and regular SDS-PAGE (lower panel) gels using anti-MBP antibody. Arrowhead and arrow indicate phosphorylated and unphosphorylated 4HA-TTG1, respectively. c, d Immunoblot analysis of phosphorylation of MBP-TTG1 variants containing single site mutations at serine or threonine by GST-SK11 (c) or GST-SK12 (d) on Phos-tag-containing (upper panels) and regular SDS-PAGE (lower panels) gels. In vitro phosphorylation of wild-type TTG1 (MBP-TTG1) with or without ATP serves as a positive or negative control, respectively. Arrowheads and arrows indicate phosphorylated and unphosphorylated MBP-TTG1, respectively. Uncropped original scans of immunoblots are shown in Supplementary Fig. 17